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Bacterial Transformation and Plasmid Purification

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Romina Garakani

on 19 December 2013

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Transcript of Bacterial Transformation and Plasmid Purification

Bacterial Transformation and Plasmid Purification
By: Romina Garakani

History of Bacterial Transformation
Transformation, conjugation, and transduction were all discovered in the last century
used to clone genes for production of thousands of proteins
have affected many things, from laundry detergents to breast cancer treatments
also used to sequence whole genomes
Transcriptional Regulation of Plasmids
Transforming Cells
Transforming Cells (cont.)
History of Plasmids
Plasmids: Structure and Function
Plasmids are extrachromosomal circles of DNA that can replicate themselves in the cytosol of bacteria
Transforming Cells (cont.)
Plasmid Purification
the next step is to grow the transformed bacteria and purify the plasmids out of the bacteria
this is referred to as a miniprep
Discovery of Bacterial Transformation
1920s- Frederick Griffith found two forms of pneumococci bacteria
1st form- encapsulated in a polysaccharide coat (referred to as Smooth, or S, strain
2nd form- nto encapsulated with a coat, referred to as Rough, or R, strain
R strain was not lethal and did not cause the disease when injected in mice
S strain killed mice
when the dead S strain bacteria mixed with live R strain and then injected in mice, they died and their blood contained live encapsulated S strains
this new bacteria was called as being "transformed"
Discovery of Bacterial Conjugation
by Joshua Lederberg and Edward Tatum
in conjugation,
E. coli
shuttle DNA across a bridge that forms between the cells
dependent on a fertility, or F, factor
only F+ bacteria can transfer DNA
Discovery of Bacterial Transduction
by Lederberg and Noron Zinder
they were testing whether salmonella bacteria could conjugate like
E. coli
they discovered that bacteria did not need to physically contact each other to transfer genetic information
indication that bacteria do not build a bridge similar to conjugation
they discovered that DNA was transferred by viruses called bacteriophages that infect bacteria
Discovery of Plasmids
by William Hayes and Lederberg
investigation of the process of bacterial conjugation lead to plasmids
it was discovered that it is not chromosomal DNA that is transferred from one cell to another, but a type of extrachromosomal DNA
Lederberg proposed the name "plasmids"
Other Discoveries of Plasmids
1961- Tsutomu Watanabe and Toshio Fukasawa were investigation
(a bacterium that was causing dysentery in Japanese hospitals)
they found that some
strains carried plasmids that conferred resistance to antibiotics
these plasmids could be transferred to non drug-resistant strains
plasmid DNA was assumed to be linear during this time
1962- Allan Campbell proposed that plasmid DNA was circular
Recombination of Plasmids and Restriction Enzymes
late 1960s- Peter Lobban introduced the concept of using restriction enzymes and plasmids to recombine DNA
1973- Stanley Cohen, Anne Chang, Herbert Boyer, and Robert Helling published a paper described how to artificially construct a biologically functional plasmid
it began the age of recombinant DNA technology
1976- Herb Boyer and Robert Swanson founded Genentech
used recombinant plasmids to produce insulin
plasmids have a starting point for replication or "ori"
ori has recognition sites for DNA polymerases that replicate DNA and enable the plasmid to be cloned as the bacterium divides
each new bacterium gets a share of the plasmids
the ori determines the copy number of plasmids, or the number of plasmids per bacterial cell
low copy number plasmids- have low numbers of plasmids in the cell and vice versa for high copy number
plasmids have genes that code for enzymes that confer resistance to antibiotics
process of if bacteria have a plasmid with an antibiotic-resistance genes, they will grow, while bacteria without it, won't grow
genes are found on either strand of the plasmid DNA
to express a gene, a plasmid must have a promoter upstream of the coding region of the gene
RNA polymerase reads in opposite directions for genes
the promoter provides a landing site of RNA polymerase so the gene can be transcribed and the protein can be made
a terminator is needed as a signal to stop transcribing the genes so that the messenger RNA is the correct length
Plasmid Uses
they are manipulated to perform two basic functions:
to express recombinant proteins (expression plasmid)
to carry or house genes that have been cloned (cloning plasmid)
genes can be inserted into a plasmid through cloning
the polymerase chain reaction isused to make copied of the gene directly from the genome of the original organism
the plasmid is then cut with restriciton enzymes to open the circle of DNA
the gene may have blunt or sticky ends from the restriciton digest
the open ends of the plasmid must be complementary to the ends
the gene is then linked, or ligated, into the plasmid using ligase
the sugar phosphate bonds are reformed between the base pairs and the plasmid again forms a circle of DNA
modern manmade pasmids are made to make cloning easier with a multiple cloning site (a string of unique restriction enzyme recognition sites)
Plasmids for Eukaryotic Expression
if plasmids are destined to be used in eukaryotic cells, they must carry features that allow them to be used in both bacteria and eukaryotes
these plasmids are called shuttle plasmids
for example, a yeast shuttle plasmid would have features that allow for replication and selection in both
E. coli
and yeast cells
the yeast shuttle would have an origin of replication and selectable marker for
E. coli
for expression in yeast, the yeast shuttle plasmid would have the sequences necessary for replication in yeast and a different selectable marker
a yeast promoter would be used to drive the gene
transcribing and translating genes takes up energy
only necessary proteins are expresd by cells at any one time
cells must regulate when and to what level they transcribe their genes
some genes need to be expressed all the time, while others need to be expressed only at certain times or in certain environments
constitutive genes- genes that are always expressed
facultative genes- genes that are transcribed only when needed
bacteria regulate expression of some of their facultative genes using operons
operons- naturally occurring control units in bacterial chromosomal DNA that consist of one promoter, multiple genes, and a single terminator
one mRNA molecular that encodes multiple proteins is produced from an operon
all the proteins encoded by the operon are ade in exactly the same proportions and made at the same time
this is beneficial for bacteria because it can instantly produce multiple proteins with a single switch for fast use of a new food source
only prokaryotes have operons
eukaryotes have more complex transcriptional reguatlion in which each gene is though to be regulated individually
to genetically engineer a cell, foreign DNA must be placed into the cell
the cell receiving the foreign DNA is tranformed
there are different ways to introduce foreign DNA into a cell
it depends on the cell type and the purpose of the experiment
the most common methods are calcium chloride transofrmation and electroporation
the success of a transformation procedure is measured by determining the transofrmation efficiency
it is calculated by quantitaing the number of bacteria that were successfully transformed per microgram of DNA
the number of transformed bacteria is determined by counting the number of colonies or colony forming units the bacteria form when spread on an agar plate
in calcium chloride transformation, actively dividing bacteria are repeatedly washed in an ice-cold calcium chloride solution
it makes the cells "chemically competent" and more permeable to DNA
chemically competent cells can be flash frozen and stored at -80 degrees for around two months
chemically competent bacteria produced in the laboratory have transformation efficiencies
to transform chemically competent bacteria, plasmid DNA is added to the cells and the mixture is incubated on ice
the cells are heat shocked in a 42 degrees Celsius bath for fifty seconds
it is then immediately placed back on ice
heat shock temporarily opens up, or fractures, the cell membranes, allowing the plasmid DNA to enter the bacterial cells
non-selective growth medium is then added to the cells
they are incubated and allowed to recover at 37 degrees Celsius
next, the cells are plated and grown overnight on a selective medium
electroporation uses eletricity to disrupt cell membranes and temporarily increase the permeability of the cells
this allows plasmid DNA to enter the cells
it can be used to transform bacteria and eukaryotic cells
eukaryotic cells can also be transformed using a method called transfection
in chemical-based transfection, plasmid DNA is first sealed in tiny oil bubles called lipid vesicles and incubated in the culture medium
the vesicles then fuse with the cell membranes and deliver the DNA into the cells
plants have cell walls that create a barrier to most delivery methods
to penetrate plant cell walls, biolistics is the method used
it uses small gold or tungsten particles coated with DNA that are shot into cells under helium pressure
a handheld device called a Helios gene gun is used for delivery of DNA into plant cells
biolistics is also used to deliver DNA into other eukaryotic cell types, including animal cells and fungi
genes can also be delivered into plants using a genetically modified Ti plasmid from
A. tumefaciens
it integrates foreign genes into the plant genome
plant cells are transformed by being infected with cultures of Agrobacterium containing genetically engineered Ti plasmids
they can be introduced into plant cells using a biolistic system
viruses can be used to deliver genes into cells
transduction is used by bacteriophages to deliver genes
scientists genetically engineer bacteriophages to encode the DNA of interest and then infect bacteria with the phage
the infected bacteria then express the recombinant DNA
baculoviruses, which infect insect larval cells, are often engineered to express recombinant genes
they are widely used to infect insect cells for recombinant protein production
retroviruses are similarly used in mammalian cells
these RNA viruses convert their RNA genome into DNA and then insert this DNA into the host cell's genome
Growing Bacteria in a Liquid Culture
to start the liquid culture, a single colony is inoculated into a culture medium containing the antibiotic
bacteria needs to be oxygenated by shaking them to hep the aerobic
E. coli
there is a lag phases where cells begin an exponential growth phase
rates of division depend on the growth conditions and the bacterial species
when the nutrients in the growth medium are consumed, the culture enters a stationary phase (number of bacteria stays the same)
finally, the culture enters the death phase, in which the number of cells decreases
cells are harvested late in the growth phase, when the bacterial number is high and the cells are still healthy and dividing
Purifying Plasmid DNA from a Culture
transformed bacteria are harvested by centrifugation, and the plasmid DNA is purified
Step 1:
the bacteria must be removed from the LB broth using a microcentrifuge
it pulls the bacteria to the bottom of the tube to form a dense mass called a pellet
the liquid becomes clear and is removed by pipetting
Step 2:
the bacteria must be broken open, or lysed, to release the cell contents, but the treatment cannot be so harsh that all the cell components are broken down
bacteria are lysed with an alkaline solution which disrupts the cell membranes an denatures proteins to release DNA from the cell
using EDTA, a chelating agent, the cell wall is weakened and the cell bursts an the alkaline conditions break the hydrogen bods thy link the strands of double-stranded DNA
it causes the double helix to unwind and remain separate
single strands stay linked together because plasmid DNA molecules are circular and supercoiled
Purifying Plasmid DNA from a Culture (cont.)
Step 3:
a low pH, high salt solution, neutralizes the alkaline pH of the lysate
the high salt concentration causes proteins to precipitate
the gDNA partially renatures and aggregates with the precipitated proteins due to its length
the plasmids reanneal into soluble, double-stranded molecules
the cellular debris is removed by centrifugation and the supernatant contains the soluble plasmid DNA
Step :
the cleared lysate has a high salt concentration that would interfere with experiments
process of purification to remove salts and other minor contaminants
it also involves concentrating the DNA into a much smaller volume
chromatography columns from commercial kits are used to purify the plasmid DNA
Based on Jim Harvey's speech structures
used to clone genes for production of thousands of proteins
have affected many things, from laundry detergents to breast cancer treatments
also used to sequence whole genomes
1961- Francois Jacob and Jacques Monod discovered the mode of action of the
it controls the production of three enzymes that are involved in the metabolism of lactose in
E. coli
operon controls the production of enzymes involved in the metabolism of another sugar, arabinose
operons are controlled by repressor proteins that are encoded by an additional gene
repressor proteins bind to the operator region near the operon and block RNA polymerase from transcribing
to turn the operon on, the sugar binds to the repressor and relieves the block, allowing transciption
the sugar is called an inducer
they can turn on genes that are normally off, while repressible operons can turn off genes that are normally on
Full transcript